Many physiological changes occur as humans age. In addition to phenotypic changes, such as change in hair color, appearance of skin, and decreased lean body mass, many changes occur at the cellular and biochemical levels. One such change is the marked decrease in telomere length as somatic cells age (Harley et al., Nature, 345:458–460 [1990]). Telomeres are highly conserved repetitive DNA sequences localized at the ends of every chromosome, which comprise tandem repeats of specific GT-rich motifs. Telomeres are necessary for proper chromosome maintenance and replication. In addition, telomeres play a role in chromosomal localization within the cell nucleus. Telomeres are essential for cell viability as they protect chromosomes from degradation and recombination.
In most organisms, telomeres are synthesized and maintained by the enzyme telomerase. Telomerase is a multisubunit ribonucleoprotein which consists of one RNA component and two protein subunits. Both the RNA and protein components are necessary for telomerase activity (See, e.g., Greider, Annu. Rev. Biochem., 65:337–365 [1996]; Greider et al., in Cellular Aging and Cell Death, Wiley-Liss Inc., New York, N.Y., pp. 123–138 [1996]). The catalytic subunit exhibits reverse transcriptase activity and utilizes the RNA template to catalyze the addition of telomeric DNA to chromosomal ends.
Most cells in adult humans do not exhibit telomerase activity; exceptions include, e.g., germ line tissues (sperm cells and oocytes) and certain blood cells (Greider et al., Cellular Aging and Cell Death, supra). Also, somatic stem or progenitor cells and activated lymphocytes exhibit telomerase activity that is typically either very low or only transiently active (Chiu et al., Stem Cells 14:239 [1996]).
There is evidence demonstrating a relationship between telomere length and cellular proliferation. Most normal somatic cells lack telomerase activity, and therefore the telomeres in these cells consistently shorten with subsequent divisions. This consistent shortening results in a finite life span for normal cells. Expression of hTERT in somatic cells will reverse the finite lifespan. (Bodner et al., Science 2: 349–352 [1998]; Vaziri et al., Curr. Biol. 8: 279–282 [1998]). The majority of immortal cell lines and tumor cells exhibit telomerase activity and the chromosomes in these cells maintain a stable telomere length as the cells divide in culture. (See Robinson & Harrington, Biotech Med., 12:6–9 [1998]). Accordingly, constitutive expression of hTERT in normal cells, which endogenously express telomerase RNA, will establish telomerase activity. These studies establish telomere maintenance, carried out by hTERT, is critical for cell survival (Oulton and Harrington, Curr. Opin. in Oncol., 12: 74–81 [2000]).
The relationship that exists between the maintenance of telomere length (i.e. telomerase activity) and cellular proliferation suggests that inhibition of telomerase activity may decrease tumor cell proliferation and provide potential cancer therapies. (Harley et al., Cold Spring Harbor Symposium on Quantitative Biology, supra; and Greider et al., Cellular Aging and Cell Death, supra.). In mouse tumor models, an increase in telomerase RNA has been shown to correlate with increased tumor progression (Blasco et al., Nature Genetics, 12:200–204 [1996]). In addition, inhibition of telomerase activity in cancer cells has been shown to cause telomere shortening, cell death and in some cases loss of the tumorigenic phenotype. (See Zhang et al., Genes Dev., 13: 2388–2399 [1999]; Hahn et al., Nat. Med. 5: 1164–1170 [1999]; Herbert et al., Proc. Natl. Acad. Sci., 96: 14276–14281 [1999]).
However, cells from telomerase RNA-deficient mice apparently lack telomerase activity, but purportedly these cells can be immortalized in culture and are able to generate tumors. See Blasco et al (Cell, 91:25–34 [1997]; see also Zakian, Cell, 91:1–3 [1997]). Further studies have indicated that late generations of mice doubly null for the INK4a (which encodes p16INKa and p19ARF) and telomerase have a lower incidence of tumor formation and their cells form fewer foci in transformation assays. (Greenberg et al, Cell, 97: 515–525 [1999]). These results demonstrate that telomerase activity or hTERT expression alone does not confer a tumorigenic phenotype. Instead these results suggest that hTERT activity in combination with other oncogenic factors, such as inactivation of the pRB/p16 pathway or expression of viral oncogenes may cause immortalization in come cell types. (See Oulton & Harrington Curr. Opin. Oncol. 12: 74–81 [1999]).
Telomerase activity has also been detected in non-malignant hyperproliferative conditions such as psoriasis and contact dermatitis. (Taylor et al., J. Invest. Dermatol. 106:759–65 [1996]; Ogoshi et al., J. Invest. Dermatol. 110:818–23, [1998]). In addition, the level of telomerase activity within these lesions does not correlate with the level of inflammation, suggesting that the detected telomerase activity is associated with cellular proliferation within the lesion and not tissue inflammation.
Shortened telomeres are postulated to result in cellular senescence by preventing or inhibiting cellular division (Harley, supra; Levy et al., J. Mol. Biol., 225:951–960 [1992]; and Harley et al., Cold Spring Harbor Symposium on Quantitative Biology, 59:307–315 [1994]). For example, the telomeres of CD28−/CD8+ T-cells, (which have a shorter life span in AIDS patients) are significantly shorter in AIDS patients as compared with the same cell-type obtained from healthy persons of the same or similar age (Effros et al., AIDS, 10: 17–22 [1996]). This study indicates that the shortened telomeres in the white blood cells of AIDS patients may be associated with the rapid senescence of these cells during the progression of the disease.
The human cDNA encoding the putative telomerase catalytic protein subunit has also been cloned (Harrington et al., Genes and Dev, 11: 3109–15 [1997]; Kilian et al., Hum. Mol. Gen., 6: 2011–9 [1997]; Meyerson et al., Cell, 90:785–795 [1997]; Nakamura et al., Science 277: 955–959 [1997]). The hTERT protein was designated hTERT by HUGO Nomenclature Committee of the Genome Database. This protein shares significant sequence similarity with the catalytic subunit of telomerase from Saccharomyces cerevisiae (EST2) (Lendvay et al., Genetics, 144:1399–1412 [1996]) Schizosaccharomyces pombe (TRT1) (Nakamura et al., Science, 277:955–959 [1997]) and Euplotes aediculatus (p123) (Linger et al., Science, 276:561–567 [1997]). hTERT mRNA has been detected in cancer cell lines and tumors which exhibit telomerase activity. Further, hTERT mRNA expression is induced upon telomerase activation, which occurs during cellular immortalization. Similarly, hTERT mRNA expression is down-regulated along with telomerase activity during human HL-60 promyelocytic leukemia cell differentiation. (Meyerson et al., Cell, 90:785–795 [1997]). The catalytic subunit is proposed to be the rate limiting determinant of telomerase activity (Nakayama et al., Nature Gen. 18: 65–8 [1998]; Weinrich et al., Nat. Gen., 17: 498–502, [1998]; Beattie et al., Curr. Biol. 8: 177–80 [1998]).
Various reports describe antibodies which bind to telomerase. WO 98/14592 describes murine and rabbit polyclonal antibodies raised against the 43 kD subunit of telomerase. Publication WO 98/14593 describes anti-hTRT (human telomerase reverse transcriptase) antibodies raised against hTRT peptides and fusion proteins. WO 99/50407 (EP 990701A1) describes monoclonal antibodies that bind to human telomerase catalytic subunit (hTERT). The antibodies include monoclonal antibodies produced by hybridoma cell lines and recombinant monoclonal antibodies. WO 99/01560 generally describes antibodies against telomerase proteins and their subunits. WO 96/19580 describes rabbit polyclonal antibodies directed towards the two polypeptide subunits, the 80 kD and 95 kD polypeptide of Tetrahymena telomerase. In addition, this reference describes use of these antibodies to screen for telomerase proteins in humans and mice. WO 99/27113 discusses in general terms the production of antibodies against mouse telomerase and its subunits. Harrington et al., Genes & Development (1997) discuss the use of hTERT (human catalytic subunit of telomerase) antisera to detect telomerase activity and to purify hTERT from HeLa cells.
Yet there is still an undeveloped need to identify new specific binding agents to telomerase. These new specific binding agents may be particularly specific for the human version of the catalytic subunit of telomerase. Preferably, the specific binding agents will be a monoclonal or polyclonal antibody or a fragment thereof, which retains the binding function of the antibody. Such binding agents will be useful in screening for diseases that are associated with telomerase activity. A class of these new specific binding agents will bind and inhibit telomerase activity; therefore providing a therapeutic for telomerase associated diseases.